The long term goal of our research program is to understand the structural basis of muscle contraction at the molecular level, i.e. how the chemical energy of ATP hydrolysis is converted into mechanical energy through a structural transformation. The benefits of the research are not limited to understanding muscle contraction, but the contractile system of muscle can serve as a prototype for many other motile processes in living cells, e.g. cell division and nutrient transport. The basic processes of muscle contraction are well understood: it is a result of cyclic interactions between the contractile proteins myosin and actin, driven by the energy of actomyosin ATP hydrolysis. To explain the muscle movement, the working hypothesis is that ATP hydrolysis brings about some structural changes in the myosin molecule while interacting with actin. However, the details of the energy conversion still remain largely unresolved. One of the obstacles is that most of the studies at the molecular level are based on isolated, in vitro systems. The link between the information obtained from the in vitro systems and the actual processes occurring intact muscle is still largely missing. The aim of our efforts is to provide such a link. X-ray diffraction from permeabilized muscle cells, the technique used in the present study, is one of the few techniques that could reveal in vivo structures in living muscle cells. Although crystal structures of myosin have revealed ligand-dependent differences (e.g. with ATP, ADP or nucleotide free), it is critical to determine if such differences occur in a fully functioning muscle cell. Diffraction data from permeabilized skeletal muscle fibers associated with each of the eight intermediate states of the actin-myosin ATP hydrolysis cycle have now been completed. The data mainly provides information on the orientations of myosin heads in the myosin filaments and when bound actin. The structures of the detached states (M.ATP, M.ADP.Pi, M.ADP, M; M=myosin) were reported by us in 1999. The strongly bound states (A.M.ADP and A.M; A=actin) have been reported by other research groups previously. In 2002, we reported data for the weakly bound states (A.M.ATP). During FY 2006, the remaining intermediate state A.M.ADP.Pi was reported, such that structural states associated with each of the eight intermediate states are now available. Briefly, the results are summarized as the following: when the myosin heads are not attached to actin, the distribution of myosin heads is ordered in a helix only when the hydrolysis products (ADP.Pi) are bound at the active site (the M.ADP.Pi state). Myosins in all other unattached states are disordered. When attached to actin in the strongly bound states (A.M.ADP and A.M), the orientation of the bound myosin heads on the actin filaments is uniform (stereospecific) and the myosin heads are immobilized. For the weakly bound A.M.ATP state, the orientation of the attached myosin heads assumes a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The state A.M.ADP.Pi hitherto has been poorly understood. This state is thought to be the critical pre-power stroke state, poised to make the transition to generate force. Hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin, structure of the ACMCADPCPi state has eluded determination either in isolated form or in muscle cells. We have succeeded in enhancing the affinity for actin in the ACMCADPCPi state. When the binding between actin and myosin was increased, unlike any other attached states, both the myosin layer lines and the actin layer lines in the diffraction pattern increased in intensity. Thus, both the myosin filaments and the actin filaments appeared to be stabilized by the attachment. We proposed that such attachment may reflect an inflexible myosin head (crossbridge) acting as a transient ?strut? between the filaments. With the close correlation found between the atomic structure, biochemistry and the filament structure, we proposed that the observed change in the mobility of the myosin heads that led to the disorder/order transformation in the filament is due to changes in the flexibility within the myosin head. A filament consisting of myosin heads with loosely coupled domains could provide a simple and straightforward explanation for the disordered distribution of the heads and their attachment to actin in a wide range of orientations. Myosin heads with reduced flexibility briefly attaching to actin may function as transient ?struts? that effectively stabilize both filaments. A stable and stiff myosin is also consistent with the concept of a ?primed? pre-power stroke state, poised to generate force. Thus a change in the compliance in the myosin head alone could be the key element in the process of force generation in muscle.